CN111732653B

CN111732653B - Monoclonal neutralizing antibody of HPV52L1 and application thereof

Info

Publication number: CN111732653B
Application number: CN201911403286.7A
Authority: CN
Inventors: 魏颖颖; 郭光华; 蔺皓; 邹国宝; 刘欣欣; 宋高尚; 沈江卫
Original assignee: Zhongsheng Fangzheng Bio Tech Co ltd
Current assignee: Zhongsheng Fangzheng Bio Tech Co ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2022-05-31
Anticipated expiration: 2039-12-30
Also published as: CN111732653A

Abstract

The invention relates to the technical field of antibody medicines, in particular to a monoclonal neutralizing antibody of HPV52L1 and application thereof. The invention provides specific monoclonal neutralizing antibodies of HPV52 and a hybridoma cell line for producing the antibodies, wherein one monoclonal neutralizing antibody with the highest neutralizing activity titer is utilized, and an indirect ELISA method, a double-antibody sandwich ELISA method, an immune hybridization method and the like are adopted, so that the monoclonal neutralizing antibodies can be widely applied to infection detection of HPV52 virus in clinical samples; the monoclonal neutralizing antibody with the highest neutralizing activity titer is used for preparing vaginal gel, vaginal cleaning solution, external washing solution, vaginal suppository, freeze-dried powder, vaginal effervescent tablets and the like, is used for treating patients infected with HPV52, and can effectively improve the clinical negative conversion rate of patients infected with HPV 52. The invention has important significance for the health development and public health prevention and control of women.

Description

Monoclonal neutralizing antibody of HPV52L1 and application thereof

Technical Field

The invention relates to the technical field of antibody medicines, in particular to a monoclonal neutralizing antibody of HPV52L1 and application thereof.

Background

Human Papilloma Virus (HPV) is a spherical double-stranded DNA virus, the diameter of virus particles is 55-60 nm, the nucleocapsid is in 20-face symmetry and consists of pentamers of 72 major capsid proteins L1 and a minor capsid protein L2. Mainly invade epithelial tissues and cells of human bodies, can cause squamous epithelial proliferation of skin mucous membranes of human bodies, and further induce various benign and malignant hyperplasia lesions. High risk HPV infections are associated with the development of various malignancies, and low risk HPV infections cause anal and genital warts.

High-risk types of HPV include 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82 and the like, which can cause Cervical Intraepithelial Neoplasia (CIN) and cervical cancer, wherein HPV16 and HPV18 are the most common oncogenic types, and monoclonal antibodies thereof are more researched, as in the Chinese patent: a monoclonal antibody against HPV 16L 1 protein, a preparation method and application thereof (publication No. CN105367652B), a monoclonal antibody capable of specifically recognizing HPV 18L 1 protein and application thereof (publication No. CN 108276491A).

Statistical data of 13.79 ten thousand people in tumor hospital of Chinese medical academy of sciences show that 7 provinces and cities are provided in Beijing, Shanghai, Jiangsu, Zhejiang, Hunan, Shanxi and Sichuan, and the detection rates of different high-risk HPV are in the following sequence: HPV16, 52, 58, 53, 39, 56, 51, 18, 59, 33, 66, 35, 31, 82, 68, 45, 26; zhejiang Taizhou hospital, based on the detection data of 3.80 ten thousand people, the detection rate sequence of high-risk HPV is as follows: HPV52, 16, 58, 39, 18, 56, 51, 33, 59, 68, 66, 31, 35, 45; the detection data of 40311 in southeast of China are HPV16 (29.9%), HPV52 (18.6%), HPV58 (17.1%), HPV18 (10.1%) and HPV33 (7.53%). As can be seen from the above data, HPV52 is second only to the common high-risk type of HPV16, but currently, the research on its monoclonal antibody is very rare, which limits the detection of HPV52 virus and the treatment of HPV52 virus using monoclonal antibodies.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a monoclonal neutralizing antibody against HPV52L1 and its application, wherein the monoclonal neutralizing antibody can be widely applied to infection detection of HPV52 virus in clinical samples, and can be used for treatment of patients infected with HPV 52.

The monoclonal antibody provided by the invention has the advantages of high specificity,

the amino acid sequence of at least one of the CDR regions of the heavy chain has the amino acid sequence shown as SEQ ID NO.1, 2 or 3 or a sequence with at least 80% sequence identity with the amino acid sequence;

wherein at least one of the CDR regions of the light chain has an amino acid sequence as set forth in SEQ ID NO 4, 5 or 6 or a sequence having at least 80% sequence identity thereto.

In the invention, the heavy chain comprises three CDR regions, and the amino acid sequences of the CDR regions respectively have the amino acid sequences shown as

SEQ ID NO

1, 2 and 3;

the light chain comprises three CDR regions, and the amino acid sequences of the CDR regions respectively have the amino acid sequences shown in SEQ ID NO. 4, 5 and 6.

Wherein the sequence shown in SEQ ID NO.1 is GFTFTSYAMH;

the sequence shown as SEQ ID NO.2 is AINPYSGSSNYPDSV;

the sequence shown in SEQ ID NO. 3 is HYSGSFFGFDY;

the sequence shown in SEQ ID NO. 4 is KSSQLYGKNVA;

the sequence shown in SEQ ID NO. 5 is WPSPRED;

the sequence shown in SEQ ID NO. 6 is QQYYSYPQT.

In the present invention, in the monoclonal antibody,

the heavy chain comprises 4 FR regions, wherein the amino acid sequence of at least one FR region has the amino acid sequence shown as SEQ ID NO. 7, 8, 9 or 10, or a sequence with at least 80 percent of sequence homology with the FR region;

the light chain comprises 4 FR regions, wherein the amino acid sequence of at least one FR region has the amino acid sequence shown as SEQ ID NO. 11, 12, 13 or 14, or a sequence with at least 80% of sequence homology with the FR region.

In the present invention, in the monoclonal antibody,

the amino acid sequences of the 4 FR regions of the heavy chain have the amino acid sequences shown in SEQ ID NO 7, 8, 9 and 10 respectively;

the amino acid sequences of the 4 FR regions of the light chain have the amino acid sequences shown as SEQ ID NOS: 11, 12, 13 and 14, respectively.

In the present invention, the sequence having at least 80% sequence homology is an amino acid sequence obtained by substituting, deleting or adding one or more amino acids from the original sequence, wherein the plurality is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32.

In the present example, the heavy chain variable region of the monoclonal antibody comprises the CDR regions shown in SEQ ID NO.1, 2 or 3 and the FR regions shown in SEQ ID NO. 7, 8, 9 and 10.

In the present example, the light chain variable region of the monoclonal antibody comprises the CDR regions shown in SEQ ID NO. 4, 5 or 6 and the FR regions shown in SEQ ID NO. 11, 12, 13 and 14.

In some embodiments, the heavy chain variable region of the monoclonal antibody comprises SEQ ID NO 7, SEQ ID NO 1, SEQ ID NO 8, SEQ ID NO 2, SEQ ID NO 9, SEQ ID NO 3, SEQ ID NO 10;

in some embodiments, the light chain variable region of the monoclonal antibody comprises SEQ ID NO 11, SEQ ID NO 4, SEQ ID NO 12, SEQ ID NO 5, SEQ ID NO 13, SEQ ID NO 6, SEQ ID NO 14, in that order.

In some embodiments, the heavy chain variable region of the monoclonal antibody has the amino acid sequence set forth in any one of SEQ ID NOs 15; the light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO 16.

In the present invention, the heavy chain constant region of the monoclonal antibody is of the IgG1 type, and the light chain constant region is of the kappa type.

In a specific embodiment, the monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC No. 19186.

The monoclonal antibody provided by the invention takes a pseudovirus particle (VLP) coated by HPV52L1 protein as immunogen, immunizes a mouse, obtains a hybridoma cell strain capable of continuously and stably secreting anti-HPV 52 by cell fusion and screening through a hybridoma technology, and is prepared. The monoclonal antibody obtained by the invention has good sensitivity and specificity, and experiments show that the monoclonal antibody and IC of HPV52VLP₉₀As low as 2ng/mL, there was no cross-reaction with the other 10 types tested (HPV6, 11, 16, 18, 31, 33, 45, 58, 59, 68). Therefore, the antibody can be applied to clinical diagnosis of HPV52 type and treatment or prevention of HPV52 type virus infection.

The invention also provides polynucleotides encoding the monoclonal antibodies or functional fragments thereof.

The functional fragment includes at least one of the CDR regions of the heavy chain or at least one of the CDR regions of the light chain.

Specifically, the polynucleotide sequence encoding the CDR region shown in SEQ ID NO.1 is shown in SEQ ID NO. 17;

the polynucleotide sequence for coding the CDR region shown in SEQ ID NO.2 is shown in SEQ ID NO. 18;

the polynucleotide sequence for coding the CDR region shown in SEQ ID NO. 3 is shown in SEQ ID NO. 19;

the polynucleotide sequence for coding the CDR region shown in SEQ ID NO. 4 is shown in SEQ ID NO. 20;

the polynucleotide sequence for coding the CDR region shown in SEQ ID NO. 5 is shown in SEQ ID NO. 21;

the polynucleotide sequence for coding the CDR region shown in SEQ ID NO. 6 is shown in SEQ ID NO. 22;

in some embodiments, the polynucleotide sequence encoding the heavy chain variable region of the monoclonal antibody provided by the invention is as set forth in SEQ ID NO: as shown at 23. The polynucleotide sequence for coding the variable region of the monoclonal antibody light chain is shown in SEQ ID NO. 24.

In some embodiments of the invention, the nucleotide sequence shown in any one of SEQ ID NOs 17 to 24 is a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 nucleotide sequences.

The invention also provides a nucleic acid vector comprising nucleotides encoding the monoclonal antibody or functional fragment thereof.

The invention also provides a recombinant host containing the nucleic acid vector.

The nucleic acid vectors of the present invention are capable of producing the antibodies expressed in vivo in a host, referred to herein as a recombinant host. The host cell is selected from escherichia coli, yeast, insect cells or mammalian cells.

The invention provides two preparation methods of the monoclonal antibody, one of which is as follows: culturing the recombinant host of the invention, and inducing the expression of the monoclonal antibody;

the second is as follows: culturing the hybridoma cell strain with the preservation number of CGMCC No.19186 to obtain the monoclonal antibody.

The invention also provides said monoclonal antibody chemically or biologically labeled.

The chemical label is an isotope, an immunotoxin and/or a chemical drug;

the biomarker is a biotin, avidin or enzyme label.

The enzyme label is preferably horseradish peroxidase or alkaline phosphatase.

The immunotoxin is preferably aflatoxin, diphtheria toxin, pseudomonas aeruginosa exotoxin, ricin, abrin, mistletoe agglutinin, modeccin, PAP, nystatin, gelonin or luffa toxin.

The monoclonal antibody is a conjugate prepared by coupling with a solid medium or a semisolid medium.

The solid phase medium or semi-solid medium refers to any support to which the monoclonal antibody or labeled monoclonal antibody of the present invention can be attached, including but not limited to nitrocellulose membrane, polyvinylidene fluoride (PVDF) membrane, iPDMS chip, microwell plate, polystyrene plate, microparticle, microcarrier, gel, etc.

The monoclonal antibody, the labeled monoclonal antibody and/or the conjugate are applied to preparation of products for detecting HPV52 type.

The HPV52 type detection product can be used as an auxiliary diagnostic tool for HPV52 virus infection, and can be a reagent combination or a kit, for example.

The research of the invention shows that the monoclonal neutralizing antibody of HPV52 provided by the invention can detect HPV52L 1/VLP/virus by adopting an indirect ELISA method, a double-antibody sandwich ELISA method or an immunoblotting method and the like.

The invention also provides a kit for detecting HPV52, which comprises the monoclonal antibody, the marked monoclonal antibody and/or the conjugate.

The invention provides a diagnosis method of HPV52 infection, which is used for detection by the kit provided by the invention.

In some embodiments, kits for indirect ELISA detection of HPV52 are provided, comprising: the monoclonal antibody of the invention. The kit also comprises an enzyme label plate, a coating solution, a confining solution, an enzyme-labeled secondary antibody, a washing solution, a luminescent substrate and a stop solution. Wherein the coating solution is 0.01M carbonate buffer solution with pH of 9.6; the blocking solution was 0.01M PBS buffer pH7.2 containing 2% BSA; the enzyme-labeled secondary antibody is goat anti-mouse IgM labeled by horseradish peroxidase; the washing solution was 0.01M PBS buffer pH7.4 containing 0.05% Tween 20; luminescent substrate TMB, stop solution is 2M H₂SO₄。

In this example, the method of diagnosis of HPV52 infection was indirect. The method specifically comprises the following steps: diluting a sample with a coating solution, coating the sample on an enzyme label plate, washing, sealing, adding the monoclonal antibody, incubating, reacting with an enzyme-labeled secondary antibody and a luminescent substrate in sequence, and determining OD (optical density) after terminating the reaction_450nmThe value is obtained.

In some embodiments, a kit for detecting HPV52 by a double-antibody sandwich ELISA method is provided, comprising: polyclonal antibodies against HPV52 VLPs and monoclonal antibodies of the invention. Further comprising: an ELISA plate, a coating solution, a confining solution, a washing solution, a luminescent substrate and a stop solution.

In this example, the diagnostic method for HPV52 infection was a double antibody sandwich ELISA.

In some embodiments, a kit for detecting HPV52 by immunoblotting is provided, wherein the kit comprises the monoclonal antibody of the invention, and further comprises a PVDF membrane, a blocking solution, TBST, an enzyme-labeled secondary antibody and ECL. Specifically, the enzyme-labeled secondary antibody is goat anti-mouse IgM labeled by horseradish peroxidase.

In this example, the diagnostic method for HPV52 infection was immunoblotting.

The monoclonal antibody, the labeled monoclonal antibody and/or the conjugate disclosed by the invention are applied to preparation of an anti-HPV 52 type virus infection preparation.

The monoclonal antibody, the marked monoclonal antibody and/or the conjugate are applied to preparation of a preparation for preventing and treating tumors and/or warts.

The tumor cell is cervical cancer, vulvar cancer, vaginal cancer, anal cancer, rectal cancer, oral cancer, tonsil cancer, penile cancer, prostatic cancer or bladder cancer;

the warts are genital warts, flat warts or common warts.

The invention also provides a medicament or vaccine comprising the monoclonal antibody, the labeled monoclonal antibody and/or the conjugate.

The preparation form of the medicine is vaginal gel, vaginal cleaning solution, external washing solution, vaginal suppository, freeze-dried powder or vaginal effervescent tablets.

In some embodiments, the pharmaceutical dosage form is a vaginal gel comprising a monoclonal antibody of the invention and an excipient comprising: disodium edetate, carbomer 974P, polycarbophil AA-1, glycerol, benzyl alcohol or triethanolamine.

In some embodiments, the pharmaceutical dosage form is vaginal irrigation solution comprising the monoclonal antibody of the invention and an excipient comprising: glycerol and physiological saline.

In some embodiments, the pharmaceutical dosage form is a topical lotion comprising the monoclonal antibody of the invention and an adjuvant comprising: glycerol, purified water, ethanol and a foaming agent. And may also include a fragrance.

In some embodiments, the pharmaceutical dosage form is a vaginal suppository comprising the monoclonal antibody of the invention and an adjuvant comprising: purified water, gelatin, glycerol and mannitol.

In some embodiments, the pharmaceutical dosage form is a lyophilized powder comprising the monoclonal antibody of the invention and an adjuvant comprising: bovine serum albumin and trehalose.

In some embodiments, the pharmaceutical dosage form is a vaginal effervescent tablet comprising the monoclonal antibody of the invention and an excipient comprising: sodium bicarbonate, ethanol solution containing 20% PEG6000, citric acid, vitamin C, lactose, microcrystalline cellulose, hydroxypropyl methylcellulose, silica gel micropowder, and magnesium stearate.

The invention also provides a method for preventing and treating HPV52 infection, and the medicine or the vaccine is administered.

The invention provides specific monoclonal neutralizing antibodies of HPV52 and a hybridoma cell line for producing the antibodies, wherein one monoclonal neutralizing antibody with the highest neutralizing activity titer is utilized, and an indirect ELISA method, a double-antibody sandwich ELISA method, an immune hybridization method and the like are adopted, so that the monoclonal neutralizing antibodies can be widely applied to infection detection of HPV52 virus in clinical samples; the monoclonal neutralizing antibody with the highest neutralizing activity titer is used for preparing vaginal gel, vaginal cleaning solution, external washing solution, vaginal suppository, freeze-dried powder, vaginal effervescent tablets and the like, is used for treating patients infected with HPV52, and can effectively improve the clinical negative conversion rate of patients infected with HPV 52. The invention has important significance for the health development and public health prevention and control of women.

Drawings

FIG. 1 shows the antibody purification effect (M: molecular weight Marker; 1: denatured antibody; 2: antibody).

Description of biological preservation

The biological material 52-1F5 is classified and named as hybridoma cell, which is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 12.5.2019, with the address of microorganism research institute of China academy of sciences No. 3, West Lu No.1, North Chen, Chaoyang, Beijing and the preservation number of CGMCC No. 19186.

Detailed Description

The invention provides a monoclonal neutralizing antibody of HPV52L1 and application thereof, and a person skilled in the art can realize the monoclonal neutralizing antibody by appropriately modifying process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. With regard to the definitions and terminology in this field, the expert can refer in particular to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

An "antibody" refers to a protein composed of one or more polypeptides that specifically bind to an antigen. One form of antibody constitutes the basic building block of an antibody. This form is a tetramer, which is composed of two identical pairs of antibody chains, each pair having a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light and heavy chains, taken together, are responsible for binding to antigen, while the constant regions are responsible for the effector functions of the antibody.

The "variable region" of an antibody heavy or light chain is the N-terminal mature region of the chain, and the amino acid composition and arrangement of the variable region varies with the specificity of the antibody. Amino acid residues in certain regions of the variable region, known as the hypervariable region, which is the site of specific binding of Ig to an antigenic determinant and is also known as the complementarity determining region, the CDR region, exhibit greater variability, while other regions of the variable region, known as the framework region, are structurally relatively stable.

The types of antibodies currently known include kappa and lambda light chains, as well as alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains or their variantsAnd equivalents thereof. Full-length immunoglobulin "light chains" (about 25kDa or about 214 amino acids) comprise a single NH domain₂A variable region of approximately 110 amino acids at the end, and a kappa or lambda constant region at the COOH-terminus. Full-length immunoglobulin "heavy chains" (about 50kDa or about 450-550 amino acids) also contain a variable region (about 116 amino acids), and one of the heavy chain constant regions, e.g., gamma (about 330 amino acids).

"antibody" includes any isotype of antibody or immunoglobulin, or antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The antibody may be labeled and detected, for example, by a radioisotope, an enzyme capable of producing a detectable substance, a fluorescent protein, biotin, or the like. The antibodies can also be bound to a solid support, including but not limited to polystyrene plates or beads, and the like.

In the present invention, the "polynucleotide" refers to a biological macromolecular compound formed by polymerizing a plurality of nucleotides, which can be ribonucleic acid or deoxyribonucleic acid and their modifications, including double-stranded or single-stranded DNA, cDNA, RNA, mRNA, etc., and can be circular or linear, or can be a part of a circular vector or a fragment in a genome.

In the present invention, the "nucleic acid vector" refers to a recombinant DNA molecule comprising a desired coding sequence and suitable nucleic acid sequences necessary for the expression of the operably linked coding gene in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotic cells include promoters, optionally including operator sequences, ribosome binding sites and possibly other sequences. Prokaryotic cells are known to utilize promoters, enhancers and termination and polyadenylation signals. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or, in some cases, the plasmid integrates into the genome. In the present specification, "plasmid" and "vector" may sometimes be used interchangeably, since plasmids are the most commonly used form of vector at present. However, the present invention is intended to include such other forms of expression vectors which serve equivalent functions, which are or will become known in the art, including, but not limited to: plasmids, phage particles, viral vectors and/or simply potential genomic inserts.

In the present invention, a "host cell" is generally a prokaryotic or eukaryotic host containing a nucleic acid vector and/or a gene of interest. Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are competent to replicate the vector encoding the protein or to express the desired protein.

The term "monoclonal antibody" refers to a preparation of antibody molecules having a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

In the invention, the method for detecting HPV52 type virus is carried out by immunology. The immunological detection is a technology for researching qualitative, quantitative or positioning of intracellular antigens (polypeptides and proteins) or antibodies by applying the principle of immunological basic principle, namely antigen-antibody reaction, namely the principle of specific combination of antigen and antibody, and developing color development agents (fluorescein, enzyme, metal ions and isotopes) for marking the antibodies through chemical reaction, wherein the technology comprises but is not limited to enzyme-linked immunoassay (indirect, direct or double antibody sandwich method), immunofluorescence, radioimmunoassay, immunoblot, immunohistochemistry, co-immunoprecipitation, chromatin co-precipitation and the like.

The medicament of the invention contains at least one functional component and also comprises a pharmaceutically acceptable carrier. The functional component comprises the antibody provided by the invention. Preferably, the pharmaceutically acceptable carrier is water, aqueous buffered solutions, isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, and the like. The type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The compositions according to the invention may comprise wetting agents, emulsifiers or buffer substances as additives.

The test materials and instruments adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 establishment of hybridoma cells

1. Animal immunization

Preparing HPV52 type L1 protein virus packaging particles by using an escherichia coli expression system, using the virus packaging particles as antigens, mixing the antigens with a Freund's complete adjuvant in equal volume for the first immunization, fully emulsifying, injecting subcutaneously by points, wherein the injection amount of each Balb/c mouse is 100 mu g each time, and immunizing 3 mice; and on the 7 th day, 14 th day and 21 th day of the first immunization, adopting emulsion of antigen and Freund's incomplete adjuvant to carry out boosting immunization, collecting blood through tail veins of mice on the 14 th day, separating serum, detecting antibody titer by using an indirect ELISA method, wherein the antibody titer of the serum of the mice is respectively 1:16000, more than 1:32000 and 1:16000, and selecting No.2 mice with highest titer for fusion.

The indirect ELISA method comprises the following steps: plate-coated with 200 ng/well of HPV52VLP, and 100. mu.L of PBS solution containing 5% skim milk powder was added to the Negative Control (NC) well, overnight at 4 ℃; discarding the liquid in the hole, washing the plate with PBS for 3 times, adding PBS solution containing 5% skimmed milk powder, sealing at 200 μ L/hole for 1 hr at room temperature; discarding the liquid in the wells, washing the plate with PBS 1 time, adding primary antibody (tail blood is diluted 2 times from 1: 500 to 1:32000 in gradient, hybridoma cell culture supernatant is diluted 1:1, mouse ascites is diluted 10 times from 1:1000 to 1:100 in gradient, purified antibody is diluted 2 times from 1 μ g/mL to 0.0005 μ g/mL), and incubating at room temperature for 1 hour; discarding the liquid in the wells, washing the plate for 3 times with PBS, adding HRP-conjugated goat anti-mouse IgG Fc (diluted 1:2000 times), and incubating for 1 hour at room temperature; washing the plate with PBS for 5 times, adding substrate A and B solution, 50 μ L/hole, reacting for 30min at room temperature in dark place; adding 1mol/L sulfuric acid to terminate the reaction, and measuring the A450 value by using a microplate reader. If the A450 value is more than 2 times larger than the negative control, the positive can be judged initially.

2. Preparation and screening of hybridoma cells

The mouse No.2 with the highest antibody titer was taken for fusion. 3 days prior to fusion, the immunogen was boosted with Freund's incomplete adjuvant. Spleen cells from mice were harvested conventionally and fused with SP2/0 cells at a 5:1 ratio with 500g/L PEG 4000. Selectively culturing with HAT culture solution, taking supernatant after 10-15 days of fusion, and screening positive hybridoma cell strains by adopting an indirect ELISA method. 268 positive cell strains are screened from 384 cell strains, 96 cell strains with higher activity are selected, after 10 generations of culture, 8 cell strains capable of stably secreting HPV52L1-VLP antibody are selected, and indirect ELISA results are shown in Table 1.

TABLE 1 Indirect ELISA test results for cell line supernatants stably secreting HPV52L1-VLP antibody

Clone number	1A8	1F5	2B3	2G12	2H6	3A11	4B7	4F9	Negative of
										A450	1.463	1.852	1.206	1.658	1.375	1.182	1.753	1.385	0.035

3. Antibody specificity detection in hybridoma cell secretory supernatants

Using indirect ELISA, the envelope antigens were HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 52, 58, 59, 68L 1VLP proteins, 100 ng/well, respectively. The detection of the antibody secreted by the 8 hybridoma cells in the cell supernatant shows that the three hybridoma cells, namely 1F5, 2G12 and 4F9, only react positively to HPV52L1-VLP protein, and the detection results of the HPV of other types are all negative, and the 3 hybridoma cells are determined to be type-specific antibodies. Combining the detection results in the table 1 and the table 2, the specific antibody 1F5 with the highest titer is selected for subsequent development.

Table 2: detection result of antibody specificity in hybridoma cell secretion supernatant

Clone number

1A8

1F5

2B3

2G12

2H6

3A11

4B7

4F9

6L1VLP

-

11L1VLP

-

16L1VLP

-

18L1VLP

-

+

-

31L1VLP

-

33L1VLP

-

+

-

45L1VLP

-

52L1VLP

+

58L1VLP

+

-

+

-

59L1VLP

-

68L1VLP

-

Example 2: preparation and characterization of monoclonal antibodies

1. Preparation of monoclonal antibody against HPV52 from ascites of mouse

Adult BALB/c mice were selected, and the hybridomas 52-1F5 were intraperitoneally inoculated at 1X 10 per mouse⁶-2×10⁶When the abdomen is obviously enlarged and the hand touches the abdomen, the skin is tense, and the 16-gauge needle can be used for collecting ascites. Ascites is centrifuged (13000r/min30 min), cell components and other precipitates are removed, supernatant is collected, antibody is purified by a ProteinG affinity column, ascites and the titer of the purified antibody are detected by an indirect ELISA method, and the result shows that the ascites is positive by 10 ten thousand times dilution and the purified antibody is still positive by 1ng/mL dilution.

2. Determination of antibody purity

The purified antibody was subjected to 12% SDS-PAGE, which showed a purity of 95% or more, and the result is shown in FIG. 1, wherein 1. the denatured antibody has two bands of light chain and heavy chain, and 2. the antibody has a single band.

3. Antibody subtype identification

The antibody subtypes produced by hybridoma cells 52-1F5 were identified by indirect ELISA using antibodies against various mouse immunoglobulin subtypes (IgG1, IgG2a, IgG2b, IgG3, IgA, IgM), and the results were indicated to be IgG1 subtypes.

4. Determination of antibody light chain and heavy chain variable region sequences

Extracting mRNA of 52-1F5 hybridoma cells, performing reverse transcription to form cDNA, performing high-fidelity PCR amplification by using variable region universal primers, inserting PCR product fragments into a T vector for DNA sequence determination, and determining the gene sequence of the variable region of 52-1F 5: the heavy chain is shown as SEQ ID NO.23, and the light chain is shown as SEQ ID NO. 24.

The obtained nucleotide sequence is translated into an amino acid sequence.

52-1F 5: the heavy chain variable region is shown as SEQ ID NO.15, and the light chain variable region is shown as SEQ ID NO. 16.

Using the sequences identified above, various genetically engineered antibodies, such as recombinant antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, diabodies, and the like, can be prepared by known antibody engineering techniques, while retaining the biological properties of the monoclonal antibody from which it is derived.

Example 3: antibody neutralization activity assay

The neutralizing activity of the antibody was tested by a pseudovirus-cell neutralization model.

Antibodies were first diluted to 1000ng/mL with PBS, then the antibodies were diluted to 0.06ng/mL with a 2-fold gradient, 50 μ L of each concentration of antibody was incubated with 50 μ L of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 52, 58, 59, 68 pseudovirus at the appropriate concentration in 96-well plates for one hour at 4 ℃, and negative serum controls, positive serum controls, cell controls, and pseudovirus controls were set. The mixtures were then added to 96-well plates previously plated with 293FT cells and incubated in a cell incubator for 72 hours. And then observing the fluorescence, collecting cells, detecting the fluorescence by using flow cytometry, calculating the fluorescence inhibition rate according to the fluorescence inhibition rate (1-experimental group/control group) multiplied by 100 percent if the fluorescence inhibition rate has the inhibition effect, and respectively taking the fluorescence inhibition rates of more than 50 percent and 90 percent as the neutralization titer of the monoclonal antibody to the pseudovirus. The result shows that the antibody only has an inhibiting effect on HPV52 pseudovirus, the 50% inhibition rate concentration is 0.5ng/mL, and the 90% inhibition rate concentration is 2 ng/mL; there was no inhibition of HPV6, 11, 16, 18, 31, 33, 45, 58, 59, 68 pseudoviruses. The antibody 52-1F5 is shown to be an HPV52 virus neutralizing antibody with high titer and good specificity.

Example 4: application of monoclonal antibody 52-1F5 in HPV52 virus diagnosis

Using the monoclonal antibody 52-1F5 obtained in example 2, indirect ELISA, double antibody sandwich ELISA, immunoblotting, etc. can be used to detect the HPV52L 1/VLP/virus content in a sample.

1. Determination of HPV52L 1/VLP/Virus content in samples by Indirect ELISA

(1) Properly diluting a detection sample in 0.01M carbonate buffer solution with pH9.6, setting multiple wells at 100 mu L/well, setting a sample diluent as a negative control well and standard substances with different concentrations as a positive control well, covering a sealing plate membrane, and incubating at room temperature for 2h or coating overnight at 4 ℃.

(2) The liquid was emptied and the residual liquid was patted dry and the plates were washed three times with 0.01M pH7.4PBS-Tween 20 wash.

(3) Blocking with 2% BSA-0.01MpH7.2 PBS for 1 hour.

(4) The plate was washed 3 times as above.

(5) Monoclonal antibody 52-1F5 was added at a 1:100 fold dilution, 0.1 mL/well, and the reaction was carried out at room temperature for 2 hours.

(6) After washing the plate 3 times, horseradish peroxidase-labeled goat anti-mouse IgM was added at 0.1 mL/well and reacted at room temperature for 1 hour.

(7) And soaking the plate in a washing solution for 5min, washing the plate for 3 times, adding a substrate (3,3',5,5' -tetramethyl benzidine, TMB) and reacting for 5-10 min at room temperature in a dark place.

(9)2M H₂SO₄The reaction was terminated and the OD value was measured at 450 nm.

(10) Based on the assay data, the HPV52L 1/VLP/virus content of the sample was determined against a standard curve.

2. Double antibody sandwich ELISA for determination of HPV52 VLP/virus content in samples

Monoclonal antibody 52-1F5 was used as a capture antibody to prepare a detection antibody that specifically recognizes HPV52 VLP/virus, and other related detection reagents known in the art were prepared to determine the HPV52 VLP/virus content of the sample by a double antibody sandwich ELISA.

The preparation method of the detection antibody capable of specifically recognizing the HPV52 VLP/virus comprises the following steps: polyclonal antibodies against HPV52 VLPs were prepared by immunizing new zealand white rabbits with HPV52 VLPs. The specific method comprises the following steps: at week 0, the primary immunization was performed by thoroughly emulsifying 500. mu.L of 0.2mg/mL HPV52VLP solution with 500. mu.L Freund's complete adjuvant to form a water-in-oil emulsion, and performing multiple dorsal subcutaneous injections to immunize 2 New Zealand white rabbits. The HPV52VLP is boosted 4 times at 2 weeks, 5 weeks, 8 weeks and 11 weeks, respectively, and each immunization is given 500. mu.L of 0.2mg/mL HPV52VLPEmulsion of the solution with 500. mu.L of Freund's incomplete adjuvant; blood sampling detection is carried out at 6 weeks, 9 weeks and 12 weeks. Indirect ELISA results showed that both new zealand white rabbits immunized with HPV52VLP 5 induced up to 2.0x10⁷And (3) high-titer antiserum with dilution times, wherein the polyclonal serum is purified and is named pAb-52 as a detection antibody in the development of a detection reagent.

By combining with other related detection reagents known in the field and by condition exploration and optimization, the kit for ELISA is constructed by using the monoclonal antibody 52-1F5 as a capture antibody and pAb-52 as a detection antibody. Through the detection of patient samples, the double-antibody sandwich ELISA detection kit is found not to identify patient samples of other HPV high-risk subtypes (such as HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82), and only specifically identifies HPV52 patient samples. Therefore, the ELISA can be used for clinical rapid detection of HPV52 infection.

3. Detection of HPV52L1 protein in sample by immunoblotting

The monoclonal antibody 52-1F5 is used as a primary antibody, Western Blotting is utilized to detect HPV52L1 protein in a sample, and the specific steps are as follows:

(1) SDS Polyacrylamide gel electrophoresis methods are described in Oseber et al, eds (molecular biology laboratory Manual) (science Press 1998). The electrophoresis is terminated by adopting 10 percent separation gel and 5 percent concentrated gel under the electrophoresis condition of 150V voltage and the distance between the bromophenol blue dye band and the bottom edge of the gel is about 1.5 cm.

(2) Electrotransfer: see e.g.Oseber et al, eds molecular biology laboratory Manual (science Press 1998). It was transferred to a PVDF (0.45 μm) membrane by electrotransfer.

(3) Immunoblotting: after the electric membrane transfer is finished, the membrane is sealed in 5% skimmed milk powder sealing solution for 1 hour at room temperature, the monoclonal antibody (1 mug/mL) prepared by the invention is used as a primary antibody, the membrane is incubated for 2 hours at room temperature or reacted overnight at 4 ℃, and the membrane is washed for 3 times (10 min each time) by TBST (TBS added with 0.5% Tween-20).

(4) And (3) using horseradish peroxidase-labeled goat anti-mouse IgM as a secondary antibody, reacting for 2h at room temperature, washing by the method, acting for 1min by ECL, and exposing and imaging in a full-automatic chemiluminescence imager (Bio-Rad Versa doc5000 MP).

Example 5 formulations for prevention or treatment of HPV52 Virus infection

The monoclonal neutralizing antibody 52-1F5 obtained in example 2 was used to prepare vaginal gel, vaginal washing solution, external washing solution, vaginal suppository, lyophilized powder, vaginal effervescent tablet, etc., for prevention and treatment of HPV infection.

1. Preparation of monoclonal neutralizing antibody 52 vaginal gel

HPV vaginal gel is prepared according to the following proportion and process: adding 0.2-0.8 wt% of edetate disodium, 0.5-1.5 wt% of carbomer 974P, 0.1-0.3 wt% of polycarbophil AA-1 and 4-8 wt% of glycerol into purified water, heating, stirring uniformly and fully swelling, adding 1-7 wt% of benzyl alcohol and 52-1F5 (the final concentration is 5ng/mL) of HPV52 monoclonal antibody when the mixture is cooled to room temperature, fully stirring uniformly, finally adding 0.6-1.0 wt% of triethanolamine, and stirring while adding to obtain homogeneous gel. 3mL of HPV vaginal gel prepared above was loaded into a disposable vaginal syringe.

The product can be used for female vagina/genital tract with positive HPV52 virus. The monoclonal neutralizing antibody 52-1F5 in the gel is specifically combined with the HPV52 virus, so that further infection of the HPV52 virus is effectively blocked, and the HPV52 virus is discharged from the body under the wrapping of carbomer gel, so that the purpose of turning negative is achieved.

2. Preparation of monoclonal neutralizing antibody 52 vaginal cleaning solution

Preparing the vaginal cleaning solution according to the following proportion and process: fully dissolving glycerol (4-8 wt percent) and HPV52 monoclonal antibody 52-1F5 (the final concentration is 10ng/mL) in physiological saline, filtering and sterilizing, and filling 3mL per sample into a disposable vagina cleaning device.

The product can be used for cleaning vagina/genital tract of female with positive HPV52 virus. The monoclonal neutralizing antibody 52-1F5 in the washing liquid is specifically combined with the HPV52 virus, so that the further infection of the HPV52 virus is effectively blocked, and the HPV52 virus is discharged out of the body, thereby achieving the purpose of negative conversion.

3. Preparation of monoclonal neutralizing antibody 52 lotion for external use

The external lotion is prepared according to the following proportion and process: fully melting glycerol (4 wt% -8 wt%) and HPV52 monoclonal antibody 52-1F5 (the final concentration is 20ng/mL) into purified water, and uniformly mixing; adding 0.1-1 wt% of rose essence into 0.5-1 wt% of ethanol for dissolving, adding 1-3 wt% of natural plant foaming agent for uniformly mixing, adding a mixed solution of glycerol/HPV 52 monoclonal antibody 52-1F5 under stirring, uniformly stirring, filtering and sterilizing. The external lotion was poured into 100mL bottles of plastic with a pump head.

The product is used for cleaning male and female genital parts in daily life, and can be applied to private parts by applying on private parts, kneading gently, and washing for a moment, so as to effectively prevent HPV52 virus infection.

4. Preparation of monoclonal neutralizing antibody 52 vaginal suppository

The pessary is prepared according to the following proportion and process: taking 300g of purified water, heating to 90 ℃, adding 200g of gelatin for fully swelling, cooling and maintaining the temperature to about 60 ℃, adding 500g of glycerol, uniformly stirring while adding, adding 600g of mannitol while stirring until the water in the glycerol gelatin matrix is evaporated to 1000g, fully dissolving, cooling to below 40 ℃, adding HPV52 monoclonal antibody 52-1F5 (the final concentration is 5ng/g), uniformly stirring at low speed, pouring into a suppository mold, standing at low temperature conventionally, taking out, and carrying out plastic packaging.

The product is used for female vagina/genital tract positive by HPV52 virus, and monoclonal neutralizing antibody 52-1F5 in suppository is specifically combined with HPV52 virus, so that further infection of HPV52 virus is effectively blocked, and HPV52 virus is discharged out of body, thereby achieving the purpose of turning negative.

5. Preparation of monoclonal neutralizing antibody 52 freeze-dried powder

The freeze-dried powder is prepared according to the following proportion and process: fully dissolving HPV52 monoclonal antibody 52-1F5 (the final concentration is 5ng/mL), bovine serum albumin (0.1 mg/mL-1 mg/mL) and trehalose (3 wt% -10 wt%) in purified water, filling 3 mL/support in a penicillin bottle, freeze-drying for 3-4 hours under the pressure of 5.0mBar, sealing and storing.

The product is used for cleaning female vagina in daily families or medical institutions, when the product is used, 3mL of normal saline is added for full dissolution, a hose is injected into the vagina for cleaning, wherein the monoclonal neutralizing antibody 52-1F5 is specifically combined with HPV52 virus, further infection of HPV52 virus is effectively blocked, and HPV52 virus is discharged out of a body, so that the aim of turning negative is achieved.

6. Preparation of monoclonal neutralizing antibody 52 vaginal effervescent tablet

Preparing the vaginal effervescent tablets according to the following proportion and process: (1) drying sodium bicarbonate (35 wt%) at 60 deg.C for 2 hr, adding appropriate amount of 20 wt% PEG6000 ethanol solution to obtain soft material, granulating with 14 mesh nylon sieve, and drying at 60 deg.C; (2) 11 wt% of citric acid, 25 wt% of vitamin C, 12 wt% of lactose and 15 wt% of microcrystalline cellulose, adding into 2% hydroxypropyl methyl cellulose alcohol solution, stirring uniformly to prepare soft materials, granulating by a 14-mesh nylon sieve, and drying at 60 ℃ for later use; (3) adding 1g of monoclonal neutralizing antibody 52-1F5 lyophilized powder, 8g of aerosil and 8g of magnesium stearate, mixing uniformly, pressing into 1000 tablets, and packaging by plastic-aluminum blister.

The product is used for vaginal/genital tract packing of women with positive HPV52 virus, is convenient to use, and the monoclonal neutralizing antibody 52-1F5 in the effervescent tablet is specifically combined with the HPV52 virus, so that further infection of the HPV52 virus is effectively blocked, and the purpose of turning negative is achieved.

Safety testing of HPV52 Virus infection treatment/prevention formulations

According to the 'disinfection technical specification' 2002 edition, the vagina mucosa irritation test is carried out on the vagina gel, vagina cleaning solution, external washing solution, vaginal suppository, freeze-dried powder and vaginal effervescent tablets prepared by the method, the experimental object is a rabbit, the irritation reaction is negative, and the safety meets the requirements.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

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Claims

1. Monoclonal antibody targeting the HPV52L1 protein,

the amino acid sequences of the CDR regions 1-3 of the heavy chain are respectively shown as SEQ ID NO 1, 2 and 3;

the amino acid sequences of CDR regions 1-3 of the light chain are respectively shown in SEQ ID NO. 4, 5 and 6.

2. The monoclonal antibody according to claim 1,

the amino acid sequences of the FR regions 1-4 of the heavy chain are respectively shown as SEQ ID NO 7, 8, 9 and 10;

the amino acid sequences of the FR regions 1-4 of the light chain are respectively shown as SEQ ID NO 11, 12, 13 and 14.

3. The monoclonal antibody according to claim 1 or 2,

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 15;

the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16.

4. The monoclonal antibody of claim 3, wherein the heavy chain constant region is of the IgG1 type and the light chain constant region is of the kappa type.

5. The monoclonal antibody according to claim 4, which is produced by a hybridoma cell line having a accession number of CGMCC No. 19186.

6. A polynucleotide encoding the monoclonal antibody or functional fragment thereof according to any one of claims 1 to 5.

7. A nucleic acid vector comprising a polynucleotide encoding the monoclonal antibody or functional fragment thereof according to any one of claims 1 to 5.

8. A recombinant host cell comprising the nucleic acid vector of claim 7.

9. A method for producing the monoclonal antibody according to any one of claims 1 to 5, comprising:

culturing the recombinant host cell of claim 8 to induce expression of the monoclonal antibody;

or culturing hybridoma cell strain with preservation number of CGMCC No.19186 to obtain the monoclonal antibody.

10. A chemically or biologically labeled monoclonal antibody according to any one of claims 1 to 5.

11. A conjugate prepared by conjugating the monoclonal antibody of any one of claims 1 to 5 or the monoclonal antibody of claim 10 to a solid medium or a semi-solid medium.

12. Use of the monoclonal antibody of any one of claims 1 to 5, the monoclonal antibody of claim 10 and/or the conjugate of claim 11 for the preparation of a product for detecting HPV type 52 virus.

13. An HPV52 virus detection kit comprising a monoclonal antibody according to any one of claims 1 to 5, a monoclonal antibody according to claim 10 and/or a conjugate according to claim 11.